The search for new compounds frequently involves screening large libraries of compounds to identify a small subset of compounds that have a desired activity or characteristic. The use of combinatorial chemistry and high-throughput screening has greatly increased the speed at which lead compounds can be identified. Indeed, such techniques have made available an increasing number of potential therapeutic agents. The challenge once such compounds have been identified is to formulate them for oral delivery. It is preferred that such therapeutic agents are amenable to oral delivery because of the decreased costs associated with this type of delivery and the clear preference of patients for oral administration. However, many agents fail at the preclinical or early clinical stage due to poor pharmacokinetics. Other potential targets for pharmaceutical agents are largely ignored due to anticipated problems of pharmaceutical agent delivery. Formulating compounds for efficient oral bioavailability has proven particularly difficult because of problems associated with uptake and susceptibility to metabolic enzymes in the intestinal tract. Delivery of compounds across the blood brain barrier or targeting compounds to specific tissues has also proven problematic.
There are two major specific transport systems of xenobiotics into and through cells: carrier-mediated systems and receptor-mediated systems. Some xenobiotics can also be taken up by passive diffusion between or through cells. Carrier-mediated systems utilize transport proteins that are anchored to the cell membrane, typically by a plurality of membrane-spanning loops and function by transporting their substrates via an energy-dependent flip-flop mechanism. Carrier-mediated transport systems are involved in the active transport of many important nutrients such as vitamins, sugars, and amino acids, as well as xenobiotic compounds. These molecules are transported by carrier-mediated systems from the lumen of the intestine into the systemic circulation or across the blood brain barrier. Carrier-mediated transporters are also present in organs such as liver and kidney, where the proteins are involved in the excretion or reabsorption of circulating compounds.
Receptor-mediated transport systems differ from the carrier-mediated systems in that rather than ferrying the substrate/ligand across the membrane, substrate binding triggers an invagination and encapsulation process that results in the formation of various transport vesicles to carry the substrate (and sometimes other molecules) into and through the cell. This process of membrane deformations that result in the internalization of certain substrates and their subsequent targeting to certain locations in the cytoplasm is referred to as endocytosis. Endocytosis encompasses several specific variations, including, for example, receptor mediated endocytosis (RME).
RME involves several defined steps beginning with the binding of a substrate to a cell-surface receptor and subsequent invagination of the membrane to form an internal vesicle variously called an early endosome, a receptosome or CURL (compartment of uncoupling receptor and ligand). In some endocytic events, after a substrate binds to its specific receptor, the substrate-receptor complex accumulates in coated pits that contain high concentrations of clathrin subunits that appear to aid in the membrane invagination process. Following internalization, the clathrin coat is lost and the pH in the endosome is lowered, thus resulting in the dissociation of the receptor-substrate complex. The endosome moves randomly or along microtubules to the trans-Golgi reticulum where the endosome is converted into one of a variety of different sorting vesicles (e.g., tubulovesicular complexes and late endosomes or multivesicular bodies). The fate of the receptor and substrate depends upon the type of sorting vesicle formed. Some ligands and receptors are recycled to the cell surface where the substrate is released and the receptor reinternalized into the membrane. In other instances, the substrate is directed to and destroyed in a lysosome, and the receptor is recycled. One type of RME is transcytosis, which refers to the process wherein an endocytotic vesicle is transported to the opposite membrane surface of a polarized cell. RME is capable of transporting a variety of compounds, including immunoglobulins, lectins, vitamins and metal ions.
A number of attempts have been made to assay or identify substrates of various transport proteins or to modify pharmaceutical agents to be improved substrates of transport proteins. Substrates so identified could be utilized to transport other compounds into or through cells. (See, e.g., Kramer et al, J. Biol. Chem. (1994), 269:10621; Mills et al, Biochim. Biophys. Acta (1992), 1126: 35; Börner et al, Eur. J. Biochem. (1998) 255: 698; Dieck et al, Glia, (1999) 25: 10; Otto et al, Am. J. Physiol. (1996) 271:C210; Abe et al, Biooconjugate Chem. (1999) 10: 24); Hussain et al, Pharm. Res. 1997, 14, 613 and McClean et al, Eur. J. Pharm. Sci. 1998, 6, 153). Some assays have involved measuring the uptake or transcellular flux of labeled compounds. Other assays have measured uptake of unlabelled compound by HPLC. However, many existing assays are tedious, are only capable of low throughput, and the delivery of many pharmaceutical agents and potential pharmaceutical agents remains to be improved. Furthermore, in assays for transcytosis and endocytosis, assays can be complicated because of the size and shape constraints imposed on assay agents by the vesicles that are part of these transport systems.
One potential approach for identifying substrates for transporter proteins is to utilize phage display technology. Phage display methods typically involve the insertion of random oligonucleotides into a phage genome such that they direct a bacterial host to express peptide libraries fused to phage coat proteins (e.g., filamentous phage pIII, pVI or pVIII). Incorporation of the fusion proteins into the mature phage coat results in the peptide encoded by the exogenous sequence being displayed on the exterior surface of the phage, while the exogenous sequence encoding the peptide resides within the phage particle. However, a significant limitation with current phage-display technology, is that it is only applicable to the display of peptides. Many of the most effective drugs, however, are small organic molecules.